Recombinant Viruses and their Use for Treatment of Atherosclerosis and Othe Forms of Coronary Artery Disease and Method, Reagent, and Kit for Evaluating Susceptibiity to Same

ABSTRACT

Recombinant viruses comprising a heterologous DNA sequence coding for a lipase involved in lipoprotein metabolism. The invention also concerns the preparation and use in therapy of said recombinant viruses, especially for the treatment or prevention of dyslipoproteinemia-related pathologies.

This application is a continuation-in-part of application Ser. No.08/737,954, filed Dec. 16, 1996, abandoned, which is a 371 ofinternational application PCT/FR95/00669, filed May 22, 1995, and acontinuation-in-part of application Ser. No. 08/817,192, filed Apr. 11,1997, which is a 371 of international application PCT/US95/13620, filedApr. 18, 1996, which is a continuation of application Ser. No.08/320,604, filed Oct. 11, 1994, issued as U.S. Pat. No. 5,658,729, eachof which is incorporated by reference herein.

The present invention relates to recombinant vectors of viral origin, totheir preparation and to their use, in particular for the treatmentand/or prevention of pathologies associated with dyslipoproteinaemias.More particularly, it relates to recombinant viruses containing a DNAsequence coding for a lipase involved in lipoprotein metabolism. Theinvention also relates to the preparation of these viral vectors, topharmaceutical compositions containing them and to their therapeuticuse, in particular, in gene therapy by which lipoprotein deficienciescan be treated. In addition, the invention relates to a method, reagentand kit for evaluating susceptibility to and causation of prematureatherosclerosis and other forms of coronary artery disease.

BACKGROUND OF THE INVENTION

Dyslipoproteinaemias are disorders of the metabolism of the lipoproteinsresponsible for transport of lipids such as cholesterol andtriglycerides in the blood and the peripheral fluids. They lead to majorpathologies associated, respectively, with hypercholesterolaemia orhypertriglyceridaemia, such as, for example, coronary artery disease.

“Coronary artery disease” is a collective term for a variety ofsymptomatic conditions including angina, myocardial infarction, andnon-specific chest, arm and face pain, which result from atherosclerosisof the arteries that supply blood to the heart.

“Premature atherosclerosis” as used herein refers to the clinicalpresentation of signs and symptoms of coronary artery disease before theage of 65.

Atherosclerosis, commonly known as “hardening of the arteries,” is acomplex disease of polygenic origin, which is defined from ahistological standpoint by deposits (lipid or fibrolipid plaques) oflipids and of other blood derivatives within the wall or endothelium ofthe large arteries (aorta, coronary arteries, carotid). These plaques,which are more or less calcified according to the degree of progressionof the process, may be coupled with lesions, and are associated with theaccumulation of fatty deposits in the arteries, consisting essentiallyof cholesterol esters.

The plaques are accompanied by a thickening of the arterial wall, withhypertrophy of the smooth muscle, appearance of foam cells andaccumulation of fibrous tissue. The atheromatous plaque protrudesmarkedly from the wall, endowing it with a stenosing characterresponsible for vascular occlusions by atheroma, thrombosis or embolismwhich occur in those patients who are most affected. Thus, thedyslipoproteinaemias can lead to very serious cardiovascular pathologiessuch as infarction, sudden death, cardiac decompensation, stroke, andthe like.

Because of the significant relationship between coronary artery diseaseand heart attacks, considerable effort has been devoted to identifyingthe biochemical causes of atherosclerosis. This research has shown thathigh levels of total cholesterol, low density lipoprotein (LDL), verylow density lipoprotein (VLDL) and triglycerides are associated withincreased risk of coronary artery disease, while high levels of highdensity lipoproteins (HDL) are associated with decreased risk ofcoronary artery disease. See, Gordon et al., The Amer. J. Med. 62:707-714 (1977). However, while observation of lipoproteins, cholesteroland triglycerides can provide a basis for identifying individuals atrisk of coronary artery disease, the levels of these substances arethemselves symptoms of an underlying biochemical defect which remainsunidentified. Thus, specific treatment of the ultimate cause rather thanan intermediate condition, and prediction of risk prior to the onset ofthis intermediate condition is not possible through such observation.

Studies directed towards the underlying cause of coronary artery diseasehave identified a number of mutations in genes coding, for proteinsinvolved in lipid transport and metabolism that appear to be associatedwith an increased risk. Examples include a large number of mutations inthe low-density lipoprotein receptor gene, Hobbs et al., Human Mutations1: 445-466 (1992), and a single mutation in the apolipoprotein-B (Apo-B)gene which underlies familial defective. Apo-B in many parts of theworld. Soria et al., Proc. Nat'l Acad. Sci. USA 86: 587-91 (1989). Inaddition, mutations in other genes which play a significant role in HDLmetabolism such as the cholesterol ester transferase protein (CETP)gene, Brown et al., Nature 342: 448-451 (1989) and the gene for Apo-A1,Rubin et al., Nature 353: 265-266 (1991), have also been shown to beassociated with either enhanced resistance or increased susceptibilityto atherosclerosis. However, these mutations are uncommon and thus farno specific mutation in any gene has been found in a significant number(i.e., >1%) of patients with coronary artery disease or prematureatherosclerosis. Accordingly, these test results while interesting donot offer the opportunity to provide evaluation or therapy tosignificant numbers of patients

At the present time these pathologies, and especially thehypercholesterolaemias, are treated essentially by means of compoundswhich act either on cholesterol biosynthesis(hydroxymethylglutarylcoenzyme A reductase inhibitors, statins), or onthe uptake and removal of biliary cholesterol (sequestering agents orresins), or alternatively on lipolysis by a mode of action which remainsto be elucidated at molecular level (fibrates). Consequently, all themajor classes of medicinal products which have been used in thisindication (sequestering agents, librates or statins) are directed onlytowards the preventive aspect of atheromatous plaque formation and not,in fact, towards the treatment of atheroma. Current treatments ofatheroma following coronary accident are merely palliative, since theydo not intervene in cholesterol homeostasis and are surgical procedures(coronary bypass, angioplasty).

SUMMARY OF THE INVENTION

It has now been found that a single point mutation in the humanlipoprotein lipase gene which results in an A-G nucleotide change atcodon 291 (nucleotide 1127) of the lipoprotein lipase gene, and asubstitution of serine for the normal asparagine in the lipoproteinlipase gene product is seen with increased frequency in patients withcoronary artery disease, and is associated with an increasedsusceptibility to coronary artery disease, including in particularpremature atherosclerosis. This is expressed as a diminished catalyticactivity of lipoprotein lipase, lower HDL-cholesterol levels and highertriglyceride levels. Thus, in accordance with one embodiment of thepresent invention there is provided a method for evaluatingsusceptibility of a human individual to premature atherosclerosis andother forms of coronary artery disease comprising the steps of:

(a) obtaining a sample of DNA from the individual; and

(b) evaluating the sample of DNA for the presence of nucleotidesencoding a serine residue as amino acid 291 of the lipoprotein lipasegene product. The presence of a serine residue is indicative ofincreased susceptibility in the patient.

The invention further provides a kit for performing the method of theinvention. Such a kit comprises a pair of primers selected to amplify aregion of a human lipoprotein lipase gene spanning amino acid 291 ofhuman lipoprotein lipase. Appropriate additional reagents may also beincluded in the kit such as polymerase enzymes, nucleoside stocksolutions and the like.

A further aspect of the present invention is a method of treatingpatients suffering from or likely to suffer from prematureatherosclerosis and other forms of coronary artery disease as a resultof a lipoprotein lipase deficiency using gene therapy. This, forexample, may be accomplished using adenovirus-mediated orretrovirus-mediated gene therapy, and can be performed using either anin vivo or an ex vivo approach.

Thus, the present invention also constitutes a novel therapeuticapproach to the treatment of pathologies associated withdyslipoproteinaernias, which may be caused by, for example, lipoproteinlipase deficiency. It proposes an advantageous solution to the drawbacksof the prior art, by demonstrating the possibility of treatingpathologies associated with dyslipoproteinaemias by gene therapy, by thetransfer and expression in vivo of a gene coding for a lipase involvedin lipoprotein metabolism. The invention thus affords a simple meanspermitting specific and effective treatment of these pathologies.

Gene therapy consists in correcting a deficiency or an abnormality(mutation, aberrant expression, and the like) or in providing for theexpression of a protein of therapeutic interest by introducing geneticinformation into the affected cell or organ. This genetic informationmay be introduced either ex vivo into a cell extracted from the organ,the modified cell then being reintroduced into the body, or directly invivo into the appropriate tissue. In this second case, differenttechniques exist, including various techniques of transfection involvingcomplexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967)891), of DNA and nuclear proteins (Kaneda et al. Science 243 (1989)375), and of DNA and lipids (Feigner et al., PNAS 84 (1987) 7413), theuse of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), andthe like. More recently, the use of viruses as vectors for gene transferhas been seen to be a promising alternative to these physicaltransfection techniques. In this connection, different viruses have beentested for their capacity to infect certain cell populations. Thisapplies especially to retroviruses (RSV, HMS, MMS, and the like), theHSV virus, adeno-associated viruses and adenoviruses.

The present invention constitutes a novel therapeutic approach to thetreatment of pathologies associated with dyslipoproteinaemias,consisting in transferring and expressing in vivo genes coding forlipases involved in lipoprotein metabolism. It is especiallyadvantageous that applicants have now shown that it is possible toconstruct recombinant viruses containing a DNA sequence coding for alipase involved in lipoprotein metabolism, and to administer theserecombinant viruses in vivo, and that this administration permits astable and effective expression of a biologically active lipase in vivo,and without a cytopathological effect.

The present invention is also the outcome of the demonstration thatadenoviruses constitute especially effective vectors for the transferand expression of such genes. In particular, the present invention showsthat the use of recombinant adenoviruses as vectors enables levels ofexpression of these genes to be obtained which are sufficiently high toproduce the desired therapeutic effect.

The present invention thus affords a novel approach to the treatment andprevention of cardiovascular and neurological pathologies associatedwith dyslipoproteinaemias.

A subject of the invention lies in a defective recombinant viruscomprising a nucleic acid sequence coding for a lipase involved inlipoprotein metabolism.

The subject of the invention is also the use of such a defectiverecombinant virus for the preparation of a pharmaceutical compositionintended for the treatment and/or prevention of cardiovascular diseases.

The present invention also relates to the use of cells modifiedgenetically in vivo or ex vivo with a virus as described above, or ofcells producing such viruses, implanted in the body, permitting asustained and effective in vivo release of a biologically active lipase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows the structure of the vector pXL2418

FIG. 2: Shows the structure of the vector pXL2419

FIG. 3: Shows the structure of the vector pXL CMV-LPL

FIG. 4: Shows the structure of the vector pXL RSV-LPL

FIG. 5: Shows the structure of the vector pXL RSV-LPLc

FIG. 6 illustrates the use of strand displacement amplification in amethod in accordance with the present invention;

FIG. 7 shows the sandwich formed when two oligonucleotide probes areused to analyze for the presence of an Asn291Ser mutation;

FIG. 8 illustrates the use of mismatch primers in accordance with theinvention to detect the Asn291Ser mutation; and

FIG. 9 shows a plasmid construct, PRc/CMV-hLPL useful in accordance withthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

Among lipases involved in lipoprotein metabolism for the purposes of theinvention, preferential mention may be made of lipoprotein lipase (LPL).

Lipoprotein lipase (LPL) is an enzyme which permits the hydrolysis oftriglycerides contained in very low density lipoproteins (VLDL) orchylomicrons. Apolipoprotein CII, which is present at the surface ofthese particles, is used as cofactor in this hydrolysis. Naturally, LPLis mainly synthesized by adipocytes in the form of a 51-kDa monomericprecursor, which is then glycosylated (58 kDa). In the blood, LPL isactive in dimeric form. Up to 80% of freshly synthesized LPL is degradedin the lysosomal compartment before being able to be secreted. After itssecretion, LPL is taken up by the luminal face of the cells of thevascular endothelium, to which it binds via glycosaminoglycans. It has avery strong affinity for heparin, which enables LPL to be displaced fromits binding site at the surface of the endothelial cell. Intravenousinjection of heparin enables LPL concentration and activity to bemeasured in patients. LPL is utilized in the vascular cells and also inliver cells as an uptake agent for lipoproteins, increasing theirretention at the cell surface and thereby promoting their uptake ortheir modification.

cDNA coding for human LPL has been cloned and sequenced (Wion et al.,Science 235 (1987) 1638-1641). Two forms of messengers coexist, of 3350and 3750 bases, mainly in adipose and muscle tissue, and originate fromthe use of 2 polyadenylation sites. They include a long untranslated 3′sequence and code for a preprotein of 475 aa, from which a leadersequence of 27 aa is cleaved to give rise to the mature monomericprotein of 448 residues. The LPL gene has also been cloned (Kirchgessneret al., Proc. Natl. Acad. Sci. USA, 1987, 262:9647-9651). The synthesis,processing and secretion of LPL are regulated in a complex manner duringdevelopment and in response to hormonal stimuli. A sizeable part of thisregulation is accomplished at transcriptional level (review in Auwerx etal., Critical Reviews in Clinical Laboratory Sciences, 1992,29:243-268).

The present invention shows that it is possible to incorporate a DNAsequence coding for LPL in a viral vector, and that these vectors enablea biologically active (dimeric) mature form of LPL to be expressed andsecreted effectively. More especially, the invention shows that in vivoexpression of active LPL may be obtained by direct administration of anadenovirus or by implantation of a cell which is productive orgenetically modified by an adenovirus or by a retrovirus incorporatingsuch a DNA.

The vectors of the invention may be used, in particular, to correct LPLdeficiencies due to mutations in the LPL gene. Such deficiencies arerelatively common and can reach an incidence of 1:5,000-1:10,000 in somepopulations (S. Santamarina-Fojo, 1992, Cur. Op. lipid., 3:186-195).These deficiencies can result from a sizeable mutation in the gene,leading to the absence of LPL synthesis or to the synthesis of atruncated or highly modified protein. The existence has, in effect, beenshown in some patients of mutations of the insertion, deletion ornonsense mutation type (review in S. Santamarina-Fojo, 1992, Cur. Op.lipid., 3:186-195). They can also result from a defect at the catabolicsite, which may be due to mutations of the missense type in the gene.They can also result from modification both at the heparin-binding siteand at the catalytic site. At the heterozygous stage, these deficienciescan represent a considerable proportion of the commonesthyperlipidaemias, including familial hypertriglycerinaemias, combinedfamilial hyperlipidaemias and postprandial hyperlipidaemias.

The present invention is especially advantageous, since it enables anexpression of LPL which is controlled and without a harmful effect to beinduced in organs which are not normally affected by the expression ofthis protein. In particular, an altogether advantageous release isobtained by implantation of cells which produce vectors of theinvention, or are infected in vivo or ex vivo with vectors of theinvention.

The lipase activity produced in the context of the present invention canbe a human or animal lipase. The nucleic acid sequence used in thecontext of the present invention can be a cDNA, a genomic DNA (gDNA), anARN (in the case of retroviruses) or a hybrid construction consisting,for example, of a cDNA into which one or more introns might be inserted.Other possible sequences are synthetic or semi-synthetic sequences. Itis especially advantageous to use a cDNA or a gDNA. In particular, theuse of a gDNA permits better expression in human cells. To permit theirincorporation in a viral vector according to the invention, thesesequences are advantageously modified, for example by site-directedmutagenesis, especially for the insertion of suitable restriction sites.The sequences described in the prior art are not, in effect, constructedfor a use according to the invention, and prior adaptations may provenecessary in order to obtain substantial expressions. In the context ofthe present invention, it is preferable to use a nucleic acid sequencecoding for a human lipase. Moreover, it is also possible to use aconstruction coding for a derivative of these lipases, especially aderivative of human LPL and HL. HL (hepatic lipase) is localized at thesurface of hepatic endothelial cells. It differs from LPL in itsinsensitivity to the activating action of apoC-II. HL is involved in thehydrolysis of IDL lipids, and also of HDL2 lipids, bringing about theirconversion to HDL3.

A derivative of these lipases comprises, for example, any sequenceobtained by mutation, deletion and/or addition relative to the nativesequence, and coding for a product retaining lipase activity. Thesemodifications may be carried out by techniques known to a person skilledin the art (see general techniques of molecular biology below). Thebiological activity of the derivatives thereby obtained may then bereadily determined, as described, in particular, in Example 3. Thederivatives for the purposes of the invention may also be obtained byhybridization from nucleic acid libraries, using the native sequence ora fragment of the latter as probe.

These derivatives are, in particular, molecules having a greateraffinity for their binding sites, molecules displaying greaterresistance to proteases, molecules having greater therapeutic efficacyor reduced side effects, or possibly novel biological properties. Thederivatives also include modified DNA sequences permitting improvedexpression in vivo.

Among preferred derivatives, there may be mentioned, more especially,natural variants, molecules in which some N- or O-glycosylation siteshave been modified or eliminated, molecules in which one or moreresidues have been substituted or molecules in which all the cysteineresidues have been substituted (muteins). There may also be mentionedderivatives obtained by deletion of regions having little or noinvolvement in the interaction with the binding sites of interest orexpressing an undesirable activity, and derivatives containingadditional residues relative to the native sequence, such as, forexample, a secretion signal and/or a junction peptide.

In a first embodiment, the present invention relates to a defectiverecombinant virus comprising a cDNA sequence coding for a lipaseinvolved in lipoprotein metabolism. In another preferred embodiment ofthe invention, the DNA sequence is a EDNA sequence.

The vectors of the invention may be prepared from different types ofvirus. Preferably, vectors derived from adenoviruses, fromadeno-associated viruses (AAV), from herpesviruses (HSV) or fromretroviruses are used. It is most especially advantageous to use anadenovirus, for a direct administration or for the ex vivo modificationof cells intended to be implanted or a retrovirus, for the implantationof productive cells.

The viruses according to the invention are defective, that is to saythey are incapable of replicating autonomously in the target cell.Generally, the genome of the defective viruses used in the context ofthe present invention hence lacks at least the sequences needed forreplication of the said virus in the infected cell. These regions may beeither removed (wholly or partially), or rendered non-functional, orsubstituted by other sequences, and in particular by the nucleic acidsequence coding for the lipase. Preferably, the defective virusnevertheless retains the sequences of its genome which are needed forencapsidation of the viral particles.

As regards adenoviruses more especially, different serotypes, thestructure and properties of which vary somewhat, have beencharacterized. Among these serotypes, it is preferable to use, in thecontext of the present invention, human adenoviruses type 2 or 5 (Ad 2or Ad 5) or adenoviruses of animal origin (see Application WO 94/26914).Among adenoviruses of animal origin which are useable in the context ofthe present invention, adenoviruses of canine, bovine, murine (e.g.:Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian oralternatively simian (e.g.: SAV) may be mentioned. Preferably, theadenovirus of animal origin is a canine adenovirus, and more preferablya CAV2 adenovirus [Manhattan or A26/61 (ATCC VR-800) strain, forexample]. It is preferable to use adenoviruses of human or canine ormixed origin in the context of the invention.

Preferably, the defective adenoviruses of the invention comprise theITRs, a sequence permitting encapsidation and the sequence coding, forthe lipase. Advantageously, in the genome of the adenoviruses of theinvention, the E1 region at least is rendered non-functional. Still morepreferably, in the genome of the adenoviruses of the invention, the E1gene and at least one of the genes E2, E4, L1-L5 are non-functional. Theviral gene of interest may be rendered non-functional by any techniqueknown to a person skilled in the art, and in particular by totalelimination, substitution, partial deletion or addition of one or morebases in the gene or genes of interest. Such modifications may beobtained in vitro (on the isolated DNA) or in situ, for example by meansof genetic engineering techniques, or alternatively by treatment bymeans of mutagenic agents. Other regions may also be modified, and inparticular the E3 (WO 95/02697), E2 (WO 94/28938), E4 (WO 94/28152, WO94/12649, WO 95/02697) and L5 (WO 95/02697) regions. According to apreferred embodiment, the adenovirus according to the inventioncomprises a deletion in the E1 and E4 regions, and the sequence codingfor LPL is inserted in the inactivated E1 region. According to anotherpreferred embodiment, it comprises a deletion in the E1 region, intowhich are inserted the E4 region and the sequence coding for LPL (see FR94/13355).

The defective recombinant adenoviruses according to the invention may beprepared by any technique known to a person skilled in the art (Levreroet al., Gene 101 (1991) 195, EP 185,573; Graham, EMBO J. 3 (1984) 2917).In particular, they may be prepared by homologous recombination betweenan adenovirus and a plasmid carrying, inter alia, the DNA sequencecoding for the lipase. Homologous recombination takes place aftercotransfection of the said adenovirus and said plasmid into a suitablecell line. The cell line used should preferably (i) be transformable bythe said elements, and (ii) contain the sequences capable ofcomplementing the portion of the genome of the defective adenovirus,preferably in integrated form in order to avoid risks of recombination.As an example of a line, there may be mentioned the human embryonickidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) whichcontains, in particular, integrated in its genome, the left-hand portionof the genome of an Ad5 adenovirus (12%), or lines capable ofcomplementing the E1 and E4 functions as are described, in particular,in Applications Nos. WO 94/26914 and WO 95/02697.

Thereafter, the adenoviruses which have multiplied are recovered andpurified according to standard techniques of molecular biology, asillustrated in the examples.

Thereafter, the adenoviruses which have multiplied are recovered andpurified according to standard techniques of molecular biology, asillustrated in the examples.

Adeno-associated viruses (AAV) are, for their part, relativelysmall-sized DNA viruses which integrate stably and in a site-specificmanner in the genome of the cells they infect. They are capable ofinfecting a broad range of cells without inducing an effect on growth,morphology or cell differentiation. Moreover, they do not appear to beimplicated in pathologies in man. The AAV genome has been cloned,sequenced and characterized. It comprises approximately 4,700 bases, andcontains at each end an inverted repeat region (ITR) of approximately145 bases, serving as origin of replication for the virus. The remainderof the genome is divided into 2 essential regions carrying theencapsidation functions: The left-hand portion of the genome, whichcontains the rep gene involved in the viral replication and expressionof the viral genes; the right-hand portion of the genome, which containsthe cap gene coding for the capsid proteins of the virus.

The use of vectors derived from AAV for the transfer of genes in vitroand in vivo has been described in the literature (see, in particular, WO91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941,EP 488,528). These applications describe different constructions derivedfrom AAV, in which the rep and/or cap genes are deleted and replaced bya gene of interest, and their use for transferring the said gene ofinterest in vitro (into cells in culture) or in vivo (directly into abody). However, none of these documents describes or suggests the use ofa recombinant AAV for the transfer and expression of a lipase ex vivo orin vivo, or the advantages of such a transfer. The defective recombinantAAVs according to the invention may be prepared by cotransfection, intoa cell line infected with a human helper virus (for encapsidation genes(rep and cap genes) of AAV. The recombinant AAVs produced are thenpurified by standard techniques.

Regarding herpes viruses and retroviruses, the construction ofrecombinant vectors has been amply described in the literature: see, inparticular, Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP178220, Bernstein et al., Genet. Eng.; 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689, and the like.

In particular, retroviruses are integrative viruses which infectdividing cells. The retrovirus genome essentially comprises two LTRs, anencapsidation sequence and three coding regions (gag, pol and env). Inthe recombinant vectors derived from retroviruses, the gag, pol and envgenes are generally deleted wholly or partially, and replaced by aheterologous nucleic acid sequence of interest. These vectors may beproduced from different types of retrovirus such as in particular,MoMuLV (Moloney murine leukaemia virus; also designated MoMLV), MSV(Moloney murine sarcoma virus), HaSV (Harvey sarcoma virus), SNV (spleennecrosis virus). RSV (Rous sarcoma virus) or alternatively Friend virus.

To construct recombinant retroviruses containing a sequence coding forLPL according to the invention, a plasmid containing, in particular, theLTRs, the encapsidation sequence and the coding sequence is generallyconstructed, and then used to transfect a so-called encapsidation cellline capable of providing in trans the retroviral functions that aredeficient in the plasmid. Generally, the encapsidation lines are capableof expressing the gag, pol and env genes. Such encapsidation lines havebeen described in the prior art, and in particular the line PA317 (U.S.Pat. No. 4,861,719), the line PsiCRIP (WO 90/02806) and the lineGP+envAm-12 (WO 89/07150). Moreover, the recombinant retroviruses cancontain modifications in the LTRs to eliminate transcriptional activity,as well as extended encapsidation sequences containing a portion of thegag gene (Bender et al., J. Virol. 61 (1987) 1639). The recombinantretroviruses produced are then purified by standard techniques.

To implement the present invention, it is most especially advantageousto use a defective recombinant adenovirus. The results given belowdemonstrate the especially advantageous properties of adenoviruses forexpressing in vivo a protein having lipase activity. The adenoviralvectors according to the invention are especially advantageous for adirect administration of a purified suspension in vivo, or for the invivo transformation of cells, in particular autologous cells, for thepurpose of their implantation. Furthermore, the adenoviral vectorsaccording to the invention possess, in addition, considerable advantagessuch as, in particular, their very high efficiency of infection,enabling infection to be carried out using small volumes of viralsuspension.

In an especially preferred embodiment, an adenovirus containing, inaddition to the gene coding for the lipase, a gene coding for anapolipoprotein is used according to the invention. The lipase ispreferably hepatic lipase and the apolipoprotein is preferably selectedfrom apoA-I and apoAIV. The two genes are advantageously used in theform of a bicistronic construction which is introduced into anadenoviral vector according to the protocol described above for theconstruction of an adenovirus containing a single gene. Advantageously,the invention relates to a recombinant adenovirus containing a genecoding for HL and a gene coding for ApoA-I, inserted into the E1 region.The adenovirus construction containing a gene coding for an apo has beendescribed in Application PCT/FR94/00422, which is incorporated herein byreference.

According to another especially advantageous embodiment of theinvention, a line is used which produces retroviral vectors containingthe sequence coding for the lipase, for implantation in vivo. The lineswhich are usable for this purpose are, in particular, PA317 (U.S. Pat.No. 4,861,719), PsiCrip (WO 90/02806) and GP+envAm-12 (U.S. Pat. No.5,278,056) cells, modified to permit the production of a retroviruscontaining a nucleic acid sequence coding for a lipase according to theinvention.

Advantageously, in the vectors of the invention, the sequence coding forthe lipase is placed under the control of signals permitting itsexpression in infected cells. These signals can be ones for homologousor heterologous expression, that is to say signals different from theones naturally responsible for the expression of the lipase. They can inparticular, be sequences responsible for the expression of otherproteins, or synthetic sequences. In particular, they can be sequencesof eukaryotic or viral genes or derived sequences, stimulating orrepressing the transcription of a gene, specifically or non-specificallyand inducibly or non-inducibly. As an example, they can be promotersequences originating from the genome of the cell which it is desired toinfect, or from the genome of a virus, and in particular the promotersof the adenovirus E1A and MLP genes, the CMV, RSV LTR promoter, and thelike. Among eukaryotic promoters, there may also be mentioned theubiquitous promoters (HPRT, vimentin, α-actin, tubulin, and the like),the promoters of intermediate filaments (desmin, neurofilaments,keratin, GFAP, and the like), the promoters of therapeutic genes (MDR,CFTR, factor VIII type, and the like), tissue-specific promoters(pyruvate kinase, villin, promoter of the fatty acid-binding intestinalprotein, α-actin promoter of smooth muscle cells, promoters specific tothe liver; Apo AI, Apo AII, human albumin, and the like) oralternatively promoters responding to a stimulus (steroid hormonereceptor, retinoic acid receptor, and the like). In addition, theseexpression sequences may be modified by adding activation, regulatory,and the like, sequences. Moreover, when the inserted gene does notcontain expression sequences, it may be inserted into the genome of thedefective virus downstream of such a sequence.

In a particular embodiment, the invention relates to a defectiverecombinant virus comprising a nucleic acid sequence coding for a lipaseinvolved in lipoprotein metabolism, under the control of a promoterchosen from RSV LTR or the CMV early promoter.

More preferably, the nucleic acid sequence used also comprises signalspermitting secretion of the lipase by infected cells. To this end, thenucleic acid sequence generally contains, upstream of the codingsequence, a signal sequence directing the lipase synthesized into thepathways of secretion of the infected cell. This signal sequence can bethe natural signal sequence of the lipase synthesized, but it can alsobe any other signal sequence which is functional in the infected cell,or an artificial signal sequence.

As stated above, the present invention also relates to any use of avirus as described above for the preparation of a pharmaceuticalcomposition intended for the treatment and/or prevention of pathologiesassociated with dyslipoproteinaemias.

The present invention also relates to a pharmaceutical compositioncomprising one or more defective recombinant viruses as are describedabove. These pharmaceutical compositions may be formulated with a viewto topical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous, intraocular, transdermal, and the like, administration.Preferably, the pharmaceutical compositions of the invention contain apharmaceutically acceptable vehicle for an injectable formulation, inparticular for an intravenous injection, such as, for example, into thepatient's portal vein. The formulations can be, in particular, isotonicsteryl solutions, or dry, in particular lyophilized, compositions which,on adding sterilized water or physiological saline, as the case may be,enable injectable solutions to be produced. Direct injection into thepatient's portal vein is advantageous, since it enables the infection tobe targeted to the liver, and thus the therapeutic effect to beconcentrated in this organ.

The doses of defective recombinant virus used for the injection may beadapted in accordance with different parameters, and in particular inaccordance with the viral vector, the mode of administration used, thepathology in question or alternatively the desired period of treatment.Generally speaking, the recombinant adenoviruses according to theinvention are formulated and administered in the form of doses ofbetween 10⁴ and 10¹⁴ pfu/ml, and preferably 10⁶ to 10¹⁰ pfu/ml. The termpfu (plaque forming unit) corresponds to the infectious power of asolution of virus, and is determined by infecting a suitable cellculture and measuring, generally after 48 hours, the number of plaquesof infected cells. The techniques of determination of the pfu titre of aviral solution are well documented in the literature.

As regards retroviruses, the compositions according to the invention cancontain the productive cells directly, with a view to theirimplantation.

In this connection, another subject of the invention relates to anymammalian cells infected with one or more defective recombinant virusesas are described above. More especially, the invention relates to anyhuman cell population infected with these viruses. The cells in questioncan be, in particular, fibroblasts, myoblasts, hepatocytes,keratinocytes, endothelial cells, glial cells, and the like.

The cells according to the invention can originate from primarycultures. They may be removed by any technique known to a person skilledin the art and then set up in culture under conditions permitting theirproliferation. As regards fibroblasts, more especially, the latter maybe readily obtained from biopsies, for example according to thetechnique described by Ham [Methods Cell. Biol. 21a (1980) 255]. Thesecells may be used directly for infection with the viruses, or stored,for example by freezing, to establish autologous banks with a view tosubsequent use. The cells according to the invention can also besecondary cultures obtained, for example, from pre-established banks(see, for example, EP 228458 EP 289034, EP 400047, EP 456640).

The cells in culture are then infected with the recombinant viruses toendow them with the capacity to produce a biologically active lipase.Infection is carried out in vitro according to techniques known to aperson skilled in the art. In particular, depending on the cell typeused and the desired number of copies of virus per-cell, a personskilled in the art can adapt the multiplicity of infection and, whereappropriate, the number of infection cycles carried out. It should beobvious that these steps must be performed under suitable conditions ofsterility when the cells are intended for administration in vivo. Thedoses of recombinant virus used for infecting the cells may be adaptedby a person skilled in the art in accordance with the desired objective.The conditions described above for in vivo administration may be appliedto infection in vitro. For infection with retroviruses, it is alsopossible to coculture the cells which it is desired to infect with cellsproducing the recombinant retroviruses according to the invention. Thismakes it possible to eliminate the need to purify the retroviruses.

Another subject of the invention relates to an implant comprisingmammalian cells infected with one or more defective recombinant virusesas are described above, or cells which produce recombinant viruses, andan extracellular matrix. Preferably, the implants according to theinvention comprise 10⁵ to 10¹⁰ cells. More preferably, they comprise 10⁶to 10⁸ cells.

More especially, in the implants of the invention, the extracellularmatrix comprises a gelling compound and, where appropriate, a supportpermitting anchorage of the cells.

For the preparation of the implants according to the invention,different types of gelling agents may be employed. The gelling agentsare used for inclusion of the cells in a matrix having the constitutionof a gel, and to promote anchorage of the cells to the support, whereappropriate. Different cellular adhesion agents may hence be used asgelling agents, such as, in particular, collagen, gelatin,glycosaminoglycans, fibronectin, lectins, and the like. Preferably,collagen is used in the context of the present invention. The collagenmay be of human, bovine or murine origin. More preferably, type Icollagen is used.

As stated above, the compositions according to the inventionadvantageously comprise a support permitting anchorage of the cells. Theterm anchorage denotes any form of biological and/or chemical and/orphysical interaction bringing about adhesion and/or binding of the cellsto the support. Moreover, the cells can either coat the support used orenter the interior of this support, or both. It is preferable, in thecontext of the invention, to use a non-toxic and/or biocompatible solidsupport. In particular, polytetrafluoroethylene (PTFE) fibres or asupport of biological origin may be used.

The implants according to the invention may be implanted in differentsites of the body. In particular, the implantation may be performed inthe peritoneal cavity, in the subcutaneous tissue (subpubic region,iliac or inguinal fossae, and the like), in an organ, a muscle, a tumouror the central nervous system, or alternatively under a mucosa. Theimplants according to the invention are especially advantageous in thatthey enable the release of the lipase in the body to be controlled: thisis, in the first place, determined by the multiplicity of infection andby the number of cells implanted. The release can then be controlledeither by withdrawing the implant, which stops the treatmentpermanently, or by the use of regulable expression systems enabling, theexpression of the therapeutic genes to be induced or repressed.

The present invention thus affords a very effective means for thetreatment or prevention of pathologies associated withdyslipoproteinaemias, especially obesity, hypertriglyceridaemia or, inthe field of cardiovascular complaints, myocardial infarction, angina,sudden death, cardiac decompensation and stroke.

In addition, this treatment can be applied equally well to man and toany animal such as sheep, cattle, domestic animals (dogs, cats, and thelike), horses, fish, and the like.

The present invention also involves detecting a mutation in the genecoding for the enzyme lipoprotein lipase in a sample of DNA obtainedfrom a patient.

The first step in the method in accordance with the invention isobtaining an appropriate sample of DNA. A suitable source of such asample is from patient blood. Isolation of the DNA from the blood can beperformed by many different methods. For example, the DNA may beisolated from the leukocytes using a salt-chloroform extraction asdescribed in Trends in Genetics 5: 391 (1989).

Once the sample of patient DNA is obtained, it may be desirable toamplify a portion of the DNA including the region of interest. Onetechnique which can be used for amplification is Polymerase ChainReaction (PCR) amplification. This technique, which is described in U.S.Pat. Nos. 4,683,202 and 4,683,195, which are incorporated herein byreference, makes use of two amplification primers each of whichhybridizes to a different one of the two strands of the DNA duplex atregions which do not overlap the site of the mutation being tested for,in this case the mutation in amino acid 291. Multiple cycles of prime),extension, and denaturation are used to produce additional copies of DNAto which the primers can hybridize. This amplification can be performedin a solution, or on a solid support (see, e.g. U.S. Pat. No. 5,200,314which is incorporated herein by reference).

The mutation site of interest is at a defined location within exon 6 ofthe lipoprotein lipase gene, the sequence of which is known in the art.Oka et al., Biochim. Biophys. Acta 1049: 21-26 (1990); Deeb et al.Biochemistry 28: 4131-4135 (1989); Wion et al., Science 235: 1638-1641(1987). Amplification primers may be used which bind to the intronregions on either side of exon 6, or which bind to portions of exon 6itself. Where amplification of the mutation site is desired, the primersshould not overlap the site of the mutation of interest. Suitableprimers include those described for exon 6 in Monsalve et al. J. Clin.Invest. 86: 728-734 (1990).

Another amplification technique which may be used in accordance with thepresent invention is known as Strand Displacement Amplification (SDA).In this technique, which is described in U.S. Pat. No. 5,270,184,incorporated herein by reference, and EP 0 497 272, and which isexemplified in FIG. 6, a gene fragment is used as the target, and aprimer is used which binds to the 3′-end of this fragment. The primer isselected to include a restriction site near its 5′-end. This can beachieved by using a primer which extends beyond the 3′-end of the targetgene fragment if there is no restriction site conveniently locatedtowards the 3′-end of the fragment from the site of interest. The primerand the target fragment (if the primer extends beyond the end of thefragment) are extended to form a duplex using modified nucleosidefeedstocks, e.g., α-thio nucleoside triphosphates, at least in theregion of the restriction cleavage site so that the newly formed strandis not susceptible to cleavage by the endonuclease. For subsequentamplification normal feedstocks are used A restriction endonuclease isintroduced which nicks the duplex at the restriction site. Extensionthen starts over at the site of the nick, at the same time that thepreviously hybridized oligonucleotide is displaced. In this as multiplecopies of one or both strands of a gene or acne fragment can beamplified without the use of temperature cycling. To use stranddisplacement amplification to amplify the mutation site responsible forthe Asn291Ser mutation, primers flanking exon 6, such as those describedin Monsalve et al. could be used.

Once amplified, the DNA may be evaluated by any of a number of methodsto determine if the Asn291Ser mutation is present. First, the amplifiedDNA can be sequenced (optionally after cloning into a TA cloning vector,available from Invitrogen, Inc.) using manual or automated sequencing ofthe amplified product. Since the complete sequence of exon 6 of normallipoprotein lipase is known, targeted sequencing primers can be readilydeveloped for this purpose.

Another approach to the detection of Asn291Ser mutations, generally usedfollowing amplification, is the use of sequence specific oligonucleotideprobes which bind to one of the mutant or wildtype form, but not to theother. Such probes generally have a length of 15 to 20 bases. Becausethe difference being evaluated is a single base, the analysis isconducted under very stringent hybridization conditions such that onlyperfect matches will form stable hybrids.

The probe used in the invention is advantageously labeled to permit itseasy detection. Suitable labels include radioactive labels, fluorescentlabels, and reactive labels such as biotin. The probe may also belabeled twice, for example with a radiolabel and a reactive label, inwhich case the reactive label may be used to the capture the DNA hybrid,for example through the reaction of biotin with an avidin-coatedsupport.

A preferred format for testing using sequence specific probes involvesthe use of a sandwich assay in which the amplified DNA is evaluatedusing two probes. The first oligonucleotide probe is either selected tobind specifically to a gene encoding a mutant human lipoprotein lipasehaving a serine residue as amino acid 291, wherein said probe binds to aportion of the gene including the bases coding for the serine residue orselected to bind specifically to a gene encoding a normal humanlipoprotein lipase having an asparagine residue as amino acid 291,wherein said probe binds to a portion of the gene including the basescoding for the asparagine residue. The second oligonucleotide probe isselected to bind to a different, non-overlapping portion of thehuman-LPL gene which is the same in both mutant and non-mutant forms.One of the two probes is labeled with a detectable label while the otheris labeled with a reactive label to facilitate immobilization. Only whenboth probes are bound to a single piece of amplified DNA will thedetectable label be immobilized through the formation of a sandwich ofthe structure shown in FIG. 2.

Various modifications of the amplification process may also be used inaccordance with the present invention to detect the presence of anAsn291Ser mutation. If intentionally mismatched primers are used duringthe amplification, the amplified nucleic acids may also be evaluated forthe presence of the Asn291Ser mutation using a technique calledrestriction fragment length polymorphism (RFLP). In order to make use ofRFLP directly to detect a point mutation (as opposed to an insertion ordeletion mutation), the mutation must result in the addition or loss ofa site cleaved by a restriction endonuclease. If this is the case, thefragments produced upon restriction endonuclease digestion of the normaland mutant gene differ in number, in size, or in both. This differencecan be detected by gel electrophoresis of the restriction fragments.

In the case of the Asn291Ser mutation, the nucleotide sequence of thecoding strand changes from

SEQ. ID. NO: 6 5′ - - - GAG ATC AAT AAA GTC - - - 3′ to SEQ. ID. NO: 75′ - - - GAG ATC AGT AAA GTC - - - 3′These fragments lack the two-fold symmetry that is associated withcleavage sites of restriction endonucleases, and thus one cannot simplyuse an enzyme which will cleave one of the sequences, but not the other.RFLP can be used, however, if a special mismatch primer is used duringthe amplification process. This primer, shown below in Example 8, bindsto the LPL gene at a site adjacent to the mutation of interest, andintroduces an intentional error into the amplified DNA. Thus, asillustrated in FIG. 8, instead of the expected sequence, the mismatchprimer produces the duplex region5′---ATAC---3′ coding strand3′---TATG---5′ non-coding strandwhen a wild-type gene is amplified, and the sequence5′---GTAC---3′ coding strand3′---CATG---5′ non-coding strandwhen a mutant gene is amplified, where the C/G pair in the fourthposition of the above fragments is the intentional mismatch. Amplifiedmutant genes therefore contain a restriction site (5′-GTAC-3′) which iscleaved by the restriction endonuclease RsaI, but amplified wild-typesequence (5′-ATAC-3′) does not. Thus, a polymorphism measurable throughrestriction fragment lengths is artificially introduced into theamplified DNA using the mismatch primers.

The amplification process may also be modified by using labeled primerswhich facilitate detection and/or capture of the amplified product. Forexample, as described in British Patent No 2 202 328, using abiotin-labeled primer as one of the two primers permits the recovery ofthe extended primers produced during the amplification reaction, e.g.,by binding the extended primers to a support coated with (strept)avidin.If the primer used is in a region flanking the mutation site, thepresence of the mutation can be detected by adding, a labeled probe,which specifically binds to the mutant or wild-type gene, to thebiotinylated amplified DNA either before or after capture of theamplified DNA on a support. If the label becomes bound to the support,this indicates that the probe was bound. Alternatively, the primer maybe one which spans the mutation site in which case amplification willoccur using a primer corresponding to the mutant sequence only when themutation is present (and vice versa). In this case, a labeled probewhich binds to a portion of the LPL gene away from the mutation site orlabeled nucleoside feedstocks may be used to introduce a label into theamplified DNA.

The presence of the Asn291Ser mutation may also be detected using acatalytic hybridization amplification system of the type described inInternational Patent Publication No. WO89/09284, which is incorporatedherein by reference. Basically, in this technique, the target nucleicacid acts as a cofactor for enzymatic cleavage of probeoligonucleotides. Thus, a substantial excess of labeled probeoligonucleotide (which binds specifically to either the mutant or thewild-type gene) is combined with the target nucleic acid under stringenthybridization conditions such that only exactly complementary strandswill hybridize to any measurable extent. An enzyme is added which willcleave the probe when it is part of a duplex, but not in single strandedform. The mixture is then cycled through multiple cycles ofannealing/enzyme digestion and denaturation. If the probe binds to thetarget, the result is the production of many small labeledprobe-fragments, and the concurrent reduction in the number of full-sizelabeled probes. Either the increase in the number of fragments or thedecrease in the number of full-sized probes can be detected and providesan amplified indication of the presence or absence of the targetsequence in the sample.

An example of an enzyme which can be used in the catalytic hybridizationamplification system is RNaseH which is used in combination with RNAprobes; which are selectively cleaved when hybridized to a strand oftarget DNA. Restriction endonucleases which do not cleavephosphorothioate-modified DNA may also be used, provided that the targetDNA is first copied to produce a phosphorothioate-modified target.Because this method combines both amplification and detection, prioramplification of the genomic DNA from the sample is generally notnecessary.

Another technique useful in the present invention which combinesamplification and detection relies on the autocatalytic replication ofcertain RNA's as described in U.S. Pat. No. 4,957,858, which isincorporated herein by reference. Briefly, in this technique areplicative RNA segment is ligated to a sequence specificoligonucleotide probe which binds to either the mutant or the wild-typeform of the Asn291Ser mutation site in exon 6 of the LPL gene. Thisligated probe is then combined with the genomic DNA in such a mannerthat the probe will bind if the matching sequence is present in thegenomic DNA, and so that unbound probe can be separated from boundprobe. For example, the genomic DNA may be immobilized on a solidsupport to facilitate washing out of unbound probe molecules.Thereafter, the RNA portion of the ligated probe is amplified, forexample using the enzyme Q-beta replicase.

Yet another form of combination amplification/detection technique whichis useful in the present invention is described in U.S. Pat. No.5,124,246 which is incorporated herein by reference. In this technique,a total of five types of oligo-nucleotide probes are used. The firsttype of probe is a multimer oligonucleotide having a “star” typeconfiguration with many generally identical arms. The second type ofprobe is a labeling probe. The labeling probe is complementary to thesequence of one of the arms of the multimer probe and includes adetectable label. The third type of probe is an immobilized probe. Aplurality of this third type of probe is affixed to a solid support. Thespecific sequences used in these first three types of probes areindependent of the nature of DNA being analyzed, except that they shouldnot hybridize with this DNA directly.

The fourth type of probe is referred to as an amplifier probe. Theseprobes are synthesized in two parts, one which is complementary to aportion of the normal sequence of exon 6 of the LPL gene away from theAsn291Ser mutation site, and one which is complementary to an arm of themultimer probe. A plurality of different types of amplifier probes isformed. These various types of probes are complementary to different,non-overlapping portions of the sequence. The fifth type of probe is acapture probe. The capture probe is also formed in two parts: one whichis complementary to the site of the Asn291Ser mutation and one which iscomplementary to the immobilized probe.

The assay is performed by combining denatured genomic DNA with theplurality of amplifier probes and capture probes under conditionspermitting hybridization. The result is the binding of numerousamplifier probes to exon 6 of the LPL gene. The capture probe will onlybind, however, if the corresponding mutant (or non-mutant, depending onthe sequence of the probe) is present. Thereafter, the solid supporthaving the third probe immobilized thereon is introduced. A solidsupport-immobilized probe-capture probe-genomic DNA-amplifier probesandwich will form if DNA complementary to the capture probe is present.The support is then washed to remove unboundmaterial, and the multimerprobe is added. The multimer probe binds to the support via theamplification probe only if the sandwich was formed in the first place.The support is then washed and a labeling probe is added. The labelingprobe will bind to all of the available arms of the multimer probe onthe solid support, thus providing numerous detectable labels for eachactual mutation site in the DNA sample.

In the foregoing discussion of amplification and detection techniques,there is frequent mention of labeled probes or labeled primers. Forpurposes of this application, the label applied to the primer may takeany form, including but not limited to radiolabels; fluorescent orfluorogenic labels; colored or chromogenic labels; chemically reactivelabels such as biotin; enzyme-labels, for example phosphatase,galactosidase or glucosidase enzymes which can produce colored orfluorescent reaction product in combination with substrates such asp-nitrophenyl phosphate (colored reaction product) or 4-methylumbelliferyl phosphate (fluorescent cleavage product); andchemiluminescent labels.

A further aspect of the present invention is the particularoligonucleotide probes which may be used in one or several of thetechniques as discussed above for detection of the Asn291Ser mutation.Thus, for use in the case of mismatch primer amplification followed byRFLP analysis there is provided an oligonucleotide primer which bindsspecifically to a gene encoding for human lipoprotein lipase in a regionadjacent to, but not overlapping the second base in the codoncorresponding to residue 291 in human lipoprotein lipase, and whichincludes a mismatched base which does not correspond to the normalsequence of human lipoprotein lipase, whereby upon extension of theprimer, using a target human lipoprotein lipase gene as a template, anextension product is produced which contains a restriction site whichcan be cleaved by a restriction endonuclease when the lipoprotein lipaseproduct made by the target gene has a serine residue as amino acid 291,and does not contain such a restriction site when the lipoprotein lipaseproduct made by the target gene has an asparagine residue as amino acid291. A preferred primer which binds to the coding strand is one in whicha base complementary to base number 1130 is changed from the normalthymine to guanine. For the non-coding strand, the change is fromadenine to cytosine. A particularly preferred mismatch primer forbinding to thecoding strand has the sequence

CTGCTTCTTT TGGCTCTGAC TGTA. SEQ. ID NO: 8.

For several of the detection methods discussed above, an oligonucleotideprobe is utilized which binds to a site which includes the site of thespecific mutation of interest. Thus, the present invention encompassestwo types of oligonucleotide probes: (1) an oligonucleotide probeselected to bind specifically to a gene encoding a mutant humanlipoprotein lipase having a serine residue as amino acid 291, whereinsaid probe binds to a portion of the gene including the bases coding forthe serine residue; and (2) an oligonucleotide probe selected to bindspecifically to a gene encoding a normal human lipoprotein lipase havinga asparagine residue as amino acid 291, wherein said probe binds to aportion of the gene including the bases coding for the asparagineresidue. These probes are preferably from 15 to 20 bases in length, andmay be selected to bind to either the coding or the non-coding strand ofthe genomic DNA. Further, the probes will advantageously include adetectable label.

A further aspect of the present invention is a kit which may be used todetect the presence of the Asn291Ser mutation. The specific componentsof the kit will depend on the nature of the evaluation being conducted.In general, however, the kit will include a pair of primers selected toamplify a region of a human lipoprotein lipase gene encoding for aminoacid 291 of human lipoprotein lipase. These primers may be primers forPCR, primers adapted for strand displacement amplification, or a normalprimer and a mismatch primer. In addition, the kit may includeoligonucleotide probes for use in the detection of the Asn291 Sermutation.

The discovery of the significance of the Asn291Ser mutation opens thedoor to the possibility of providing gene therapy to individuals havingthis mutation and thus to prevent or delay the onset of coronary arterydisease and particularly premature atherosclerosis. In addition, sincegene therapy to correct this defect would provide a patient with a fullyfunctional lipoprotein lipase enzyme, therapeutic agents and methodsused for this purpose may also be used effectively for other conditionsassociated with LPL mutations. Such conditions include infantile failureto thrive, hepatosplenomegaly, eruptive xanthomas, chronic and/orepisodic abdominal pain, pancreatitis and lactescent plasma due to anaccumulation of chylomicrons and very low density lipoproteins or theirremnants in the plasma.

Gene therapy to introduce functional LPL may reduce the clinicalmanifestations stemming from hypenriglyceridemia in both LPL deficienthomozygotes and heterozygotes. This gene transfer can be accomplished,as previously described, using adenovirus-DNA-polylysine conjugates;adenovirus constructs in which the normal LPL gene is inserted into theviral genome; or retroviral constructs in which the normal LPL gene isinserted into the viral genome. The vector may be introduced directly,for example by parenteral injection into the patient, or may beintroduced via an implanted pseudo-organ.

FIG. 9 shows a plasmid construct useful in accordance with the presentinvention. As shown, the plasmid pRc/CMV-hLPL is 7.90 Kbases in size.The preparation of this particular plasmid is described below in Example9. It will be appreciated by persons skilled in the art, however, thatvariations in this technique, or the precise structure of the plasmidmay be made without departing from the present invention provided thatthe plasmid contains a functional h-LPL gene and an appropriatepromoter. For example, tissue-specific promoters, particularly adiposetissue specific or muscle specific promoters might be used in place ofthe CMV promoter. Furthermore, while the SV40 promoter and theantibiotic resistance markers are convenient for research purposes, theyare not necessary for therapeutic purposes.

To prepare a plasmid for transfection into mammalian, and particularlyhuman cells, the plasmid is complexed with an adenovirus-polylysineconjugate. In general this process involves the harvesting andpurification of a suitable adenovirus, for example a virus which isincompetent as a result of an E1A or an E3 deletion mutation. Thepurified virus is then conjugated with a polycationic material forassociating with DNA such as polylysine, polyarginine or protamine, forexample using a bifunctional reagent such as ethyl-3,3-dimethylaminopropyl carbodiimide (EDC) as a crosslinking agent. When theresulting adenovirus-polylysine conjugate is combined with a plasmidcontaining a normal LPL gene, an adenovirus-DNA-polylysine complex formsspontaneously. This complex transfects mammalian cells of various typeswhen placed in media with the cells with relatively high efficiency, andthe transfected cells produce functional LPL.

Mammalian cells may also be transduced (or transfected) using anadenovirus into which a gene encoding for normal LPL has been inserted.Preferred adenoviruses are those with an E1 or an E3 deletion mutationrendering the virus incompetent. The h-LPL gene can be convenientlyinserted into the virus at the site of the deletion.

Specific modified adenoviruses useful in the present technique are basedon the RSV β-Gal adenovector described by Stratford-Perricaudet et al.,J. Clin. Invest. 90: 626-630 (1990). This adenovector is based onadenovirus Ad5. Human LPL cDNA is introduced into the vector byhomologous recombination using a modified form ofStrafford-Perricaudet's pLTRβGalpIX plasmid. The plasmid contains an RSVLTR promoter or a CMV plus intron promoter, human LPL cDNA, a poly Asite plus small intron from SV40 derived from a pSV2 vector. Mulligan etal., Science 209: 1422-1427 (1980) which are inserted betweennucleotides 455 to 3329 of an Ad5 DNA which is also deleted in the E3region. This results in the deletion of E1A and part of E1B, but, leavespIX intact. The resulting adenoviruses are non-replicating but can bepropagated in 293 cells which transcomplement the E1A activity.

A third type of vector which may be used to transduce (or transfect)mammalian cells is a retroviral vector. Suitable vectors includemyeloproliferative sarcoma virus (MPSV)-based retroviral vectors intowhich human LPL cDNA is inserted under the transcriptional control ofthe constitutive enhancer-promoter regulatory elements of the MPSV longterminal repeat (LTR).

Gene transfer vectors can be introduced into a human subject either invivo or ex vivo. In the case of an in vivo treatment, the gene transfervector may be simply injected into the patient, for exampleparenterally, and allowed to find suitable target cells. In the case ofex vivo treatment, cells are grown in vitro and transduced ortransfected with the virus, embedded in a carrier such as a collagenmatrix, which is then implanted in the patient, for example as asub-cutaneous implant. Preferred cells for use in ex vivo applicationsare fibroblast cells taken from the patient who will receive theimplant.

General Techniques of Molecular Biology

The methods traditionally used in molecular biology, such as preparativeextractions of plasmid DNA, centrifugation of plasmid DNA in a caesiumchloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, phenol orphenol/chloroform extraction of proteins, ethanol or isopropanolprecipitation of DNA in a saline medium, transformation in Escherichiacoli, and the like, are well known to a person skilled in the art andare amply described in the literature [Maniatis T. et al., “MolecularCloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New. York, 1987].

Plasmids of the pBR322 and pUC type and phages of the M13 series are ofcommercial origin (Bethesda Research Laboratories).

To carry out ligation, the DNA fragments may be separated according totheir size by agarose or acrylamide gel electrophoresis, extracted withphenol or with a phenol/chloroform mixture, precipitated with ethanoland then incubated in the presence of phage T4 DNA ligase (Biolabs)according to the supplier's recommendations.

The filling in of 5′ protruding ends may be performed with the Klenowfragment of E. coli DNA polymerase I (Biolabs) according to thesupplier's specifications. The destruction of 3′ protruding ends isperformed in the presence of phage T4 DNA polymerase (Biolabs) usedaccording to the manufacturer's recommendations. The destruction of 5′protruding ends is performed by a controlled treatment with S1 nuclease.

Mutagenesis directed in vitro by synthetic oligodeoxynucleotides may beperformed according to the method developed by Taylor et al. [NucleicAcids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.

The enzymatic amplification of DNA fragments by the so-called PCR[Polymerase-catalysed Chain Reaction, Saiki R. K. et al., Science 230(1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155(1987) 335-350] technique may be performed using a DNA thermal cycler(Perkin Elmer Cetus) according to the manufacturer's specifications.

The verification of the nucleotide sequences may be performed by themethod developed by Sanger et al. [Proc. Natl. Acad. Sci. USA. 74 (1977)5463-5467] using the kit distributed by Amersham.

The present invention will be described more completely by means of theexamples that follow, which one should consider as illustrative andnon-limiting.

Example 1

Construction of the Vector pXL2418 Carrying the Gene Coding for LPLUnder the Control of the Cytomegalovirus (CMV) Early Promoter (FIG. 1)

This example describes the construction of a vector comprising a cDNAsequence coding for LPL, under the control of a promoter consisting ofthe cytomegalovirus (CMV) early promoter, as well as a region of the Ad5adenovirus genome permitting homologous recombination. This vector wasconstructed as described below.

1.1. Construction of the Vector pXL2375

The vector pXL2375 contains, in particular, a region of the Ad5adenovirus genome and a DNA sequence coding for apolipoprotein AI underthe control of the CMV promoter. More especially, the CMV promoter usedextends as far as the donor 5′ splicing site linked to the 107 bpnearest the 3′ end of the synthetic intron described by O'Gorman et al.(Science 251 (1991) 1351). The construction of this vector has beendescribed in detail in copending Application FR 93/05125. It isunderstood that similar constructions may be carried out by a personskilled in the art.

1.2. Construction of the cDNA Sequence Coding for LPL

Plasmid pHLPL 26-1 described by Wion et al. (Science 235 (1987)1638-1641) contains an incomplete sequence of LPL cDNA. Thus, thisplasmid contains bases 272 to 1623 of LPL cDNA flanked by two EcoRIsites, cloned to the EcoRI site of a plasmid p′GEM1 (Promega).

The EcoRI fragment of pHLPL 26-1 containing the partial LPL cDNA wasrecloned into the EcoRI site of a plasmid pMTL22 (Chambers et al., Gene,1988, 68:138-149), in the orientation placing the 5′ bases of the cDNAon the same side as the BglII site of pMTL22. The resulting plasmid wascalled pXL2402.

The RNA of human adipose tissues was then extracted according to thetechnique of Chromczynski and Sacchin (Anal. Biochem. 162 (1987)156-159). From this RNA preparation, an amplification was carried out byRT-PCR so as to isolate the missing portion of the LPL cDNA. To thisend, the following primers were used:

Sq4541: (SEQ ID NO: 1) TTA GAT CTA TCG ATA GAT GGA GAG CAA AGC CCC  TGThis primer makes it possible to introduce a BglII site as well as aClaI site upstream of the ATG.

Sq3810: (SEQ ID NO: 2) TAC ATT CCT GTT ACC GTC CAG CCA TGG ATC

The 260-bp PCR fragment obtained after 25 amplification cycles was thencloned into plasmid pCR-II (Invitrogen) and sequenced for verification.It was then introduced via the BglII and NcoI sites into the vectorpXL2402, which reconstitutes a complete cDNA preceded by a ClaI site andfollowed by a SalI site. The resulting plasmid was called pXL2417.

1.3. Construction of the Vector pXL2418.

Lastly, the LPL cDNA was inserted into plasmid pXL2375 between the Sailand ClaI sites, following excision of the Apo A1 cDNA with these sametwo enzymes. The plasmid obtained was designated pXL2418 (FIG. 1).

Example 2

Construction of the Vector pXL2419 Carrying the Gene Coding for LPLUnder the Control of the Promoter of the Rous Sarcoma Virus LTR (RSVLTR) (FIG. 2)

This example describes the construction of a vector comprising a cDNAsequence coding for LPL, under the control of a promoter consisting ofthe Rous sarcoma virus LTR (RSV LTR), as well as a region of the Ad5adenovirus genome permitting homologous recombination. This vector wasconstructed as described below.

2.1. Construction of the Vector pXL2244.

The vector pXL2244 contains, in particular, a region of the Ad5adenovirus genome and a DNA sequence coding for apolipoprotein AI underthe control of the RSV LTR promoter.

2.2. Construction of a cDNA Sequence Coding for LPL.

The cDNA sequence coding for LPL used in this example is that describedin Example 1.2.

2.3. Construction of the Vector pXL2419.

The LPL cDNA was inserted into plasmid pXL2244 between the SalI and ClaIsites, following excision of the Apo A1 cDNA with these same twoenzymes. The plasmid obtained was designated pXL2419 (FIG. 2).

Example 3 Construction of the Vectors pXL RSV-LPL and pXL CMV-LPL

The vector pRC-CMV LPL contains a fragment of LPL cDNA extending frombases 1 to 2388 of the sequence published by Wion et al., cloned at theHindIII and XbaI sites of the expression vector pRC-CMV (Invitrogen).The HindIII site was modified to a ClaI site by inserting theoligonucleotide AGC TAC ATC GAT GT (SEQ ID NO: 3). The LPL cDNA and thepolyadenylation site of bovine growth hormone (initially contained inpRC-CMV) are finally extracted from the pRCMV-LPL by SphI cleavage,treatment with T4 polymerase and ClaI cleavage. The fragment therebyobtained was cloned into the vectors pXL2418 (Example 1) and pXL2419(Example 2) cut with ClaI and EcoRV, generating the vectors pXL CMV-LPL(FIG. 3) and pXL RSV-LPL (FIG. 4), respectively.

Example 4 Construction of the Vector pXL RSV-LPLc

This example describes the construction of a vector which is usable togenerate recombinant viruses containing a short cDNA coding for LPL.

A shorter cDNA (bases 146 to 1635 of the sequence of Wion et al.) wascloned from the RNA of human adipose tissue. The primers ATC GGA TCC ATCGAT GCA GCT CCT CCA GAG GGA CGC (SEQ ID NO: 4) and ATC TCT AGA GTC GACATG CCG TTC TTT OTT CTG TAG (SEQ ID NO 5), which create, respectively, aBamHI site and a CalI site at the 5′ end of the cDNA, as well as an XbaIsite and a SalI site at the 3′ end of the LPL cDNA, were used.

This PCR fragment was cloned into PCR II, and its sequence verified inits entirety. The LPL cDNA was then released via the BamHI and XbaIsites and cloned into an expression vector pcDNA3 (Invitrogen) forverification of the expression, generating plasmid pcDNA3-LPLc.

The ClaI-SalI fragment containing the LPL cDNA was finally cloned at thesame sites into plasmid pXL RSV-LPL (Example 3) to generate the shuttleplasmid pXL RSV-LPLc (FIG. 5).

Example 5 Functionality of the Vectors of the Invention: Demonstrationof an LPL Activity

The capacity of the vectors of the invention to express a biologicallyactive form of LPL in a cell culture was demonstrated by transienttransfection of 293 CosI cells. To this end, cells (2×10⁶ cells per dish10 cm in diameter) were transfected (8 μg of vector) in the presence ofTransfectam. The expression of the sequence coding for LPL andproduction of a biologically active protein were demonstrated either interms of mass using an immunoenzymatic test (5.1.), or in terms oflipase activity (5.2.).

5.1. Measurement of LPL in Terms of Mass.

An Immulon I ELISA plate (Dynatech) was coated with anti-bovine LPLmonoclonal antibodies cross-reacting with human LPL (20 μg/ml in PBS, 50μl/well). The potential sites remaining in the wells were then blocked(saturated) by incubation in the presence of 1% gelatin for 1 hour atroom temperature. The samples to be measured were then incubated for 1hour at 37° C.

Visualization was carried out with an anti-LPL serum diluted to 10μg/ml, 100 μl/well, followed by a peroxidase-labelled antiserum.Peroxidase activity was detected using a TMB substrate (Kirkegaard andPerry Laboratories Inc. kit) and reading of the absorbance at 490 nm.

5.2. Measurement of LPL Activity.

Total lipase activity was measured on a substrate composed of anemulsion of 0.3 mg of triolein (Sigma), 75nCi oftri(1-¹⁴C)oleoylglycerol (55mCi/mmol, Amersham), 18 mg of BSA (FractionV, Sigma) and 25 μl of normal human plasma as a source of ApoCII, allthese constituents in a final volume of 500 μl of 0.223M Tris pH 8.5.Generally speaking, activity was measured on 100 μl of supernatant oftransfected cells or 50 μl of post-heparin plasma.

After 1 hour of incubation at 37° C., the reaction was stopped by adding3.25 ml of extraction buffer (chloroform/methanol/heptane, 10:9:7 v/v/v)and 0.75 ml of carbonate/borate buffer pH 10.5, and the organic phasecounted to determine the amount of fatty acids liberated.

To determine the activity specifically associated with LPL, themeasurement of hepatic lipase activity was carried out in the presenceof a 1M concentration of NaCl (which inhibits LPL), and then subtractedfrom the total activity. It was also possible to inhibit lipoproteinlipase activity with a specific monoclonal antibody (Babirak et al.Atheriosclerosis, 1989, 9:326-334).

Plasmids pXL RSV-LPL and pXL CMV-LPL were tested by transfection intoCosI cells by comparison with plasmid pRC CMV-LPL. The results arepresented in Table 1.

TABLE 1 Activity in the Expression supernatant vector Day 1 pRC-CMV-LPL24.5 pXL RSV-LPL 15.1 pXL CMV-LPL 22.9

Plasmid pcDNA-LPLc was tested by transfection into 293 cells bycomparison with an expression vector pcDNA3 containing the same cDNA asthe vector pRC-CMV-LPL. The results are presented in Table 2.

TABLE 2 Activity in the Activity in the Expression supernatantsupernatant vector Day 1 Day 2 pcDNA3-LPL 106.4 mU/ml 106.7 mU/mlpcDNA3-LPLc 114 mU/ml 109.6 mU/ml

Example 6 Construction of Recombinant Adenovirus Ad-CMV.LPL Containing aSequence Coding for LPL Lipase

The plasmids prepared in Examples 1 to 4 were linearized andcotransfected for recombination with the deficient adenoviral vector, inhelper cells (line 293) providing in trans the functions encoded by theE1 (E1A and E11B) regions of adenovirus.

More especially, the adenovirus Ad.CMV.LPL was obtained by homologousrecombination in vivo between the adenovirus Ad.RSVβgal(Stratford-Perricaudet et al. J. Clin. Invest 90 (1992) 626) and plasmidpXL2418 or pXL CMV-LPL according to the following protocol: thelinearized plasmid pXL2418 or pXL CMV-LPL and the adenovirus labelledAd.RSVβgal linearized with ClaI were cotransfected into line 293 in thepresence of calcium phosphate to permit homologous recombination. Therecombinant adenoviruses thus generated were selected by plaquepurification. After isolation, the recombinant adenovirus was amplifiedin the cell line 293, yielding a culture supernatant containing theunpurified recombinant defective adenovirus having a titre ofapproximately 10¹⁰ pfu/ml.

The viral particles were then purified by centrifugation on a caesiumchloride gradient according to known techniques (see, in particular,Graham et al., Virology 52 (1973) 456). The adenovirus Ad-CMV.LPL werestored at −80° C. in 20% glycerol.

The same protocol was reproduced with plasmid pXL2419 or pXL RSV-LPL orpXL RSV-LPLc, yielding the recombinant adenovirus Ad.RSV.LPL orAd.RSV.LPLc.

Example 7 In Vivo Transfer of the LPL Gene by a Recombinant Adenovirus

This example describes the transfer of the LPL gene in vivo by means ofan adenoviral vector according to the invention.

The adenoviruses injected were the adenoviruses Ad-CMV.LPL andAd.LTR.LPL prepared in Example 5, used in purified form (3.5×10⁶pfU/μl), in saline phosphate solution (PBS). These viruses were injectedinto C57B1/6 mice intravenously using the tail Vein, the retro-orbitalsinus or the portal vein. The expression of an active form of LPL wasdemonstrated under the conditions described in Example 5.

Example 8

The significance of the mutation resulting in a serine in place of anasparagine as amino acid 291 in human lipoprotein lipase (“Asn291Sermutation”) was discovered as a result of a case controlled study of alarge homogeneous sample of patients undergoing diagnostic coronaryangiography. A total of 807 men, all of whom were of Dutch descent andhad angiographically proven atherosclerosis with more than 50% stenosisof at least one major coronary vessel were included in the study. All ofthe patients were less than 70 years of age, and had total cholesterollevels between 4 and 8 mmol/l and triglyceride levels which did notexceed 4 mmol/l. The control group for the study included 157 personswho did not have any history of angina or premature atherosclerosis, andwho exhibited no signs of vascular disease upon physical examination.The controls were all less than 60 years of age and had baseline HDLlevels greater than 0.95 mmol/l and triglyceride levels of less than 2.3mmol/l.

DNA was extracted from leukocytes using a salt-chloroform extractionmethod as described in Trends in Genetics 5: 391 (1989). Exon 6 of theLPL gene was amplified with a 5′-PCR primer located in intron 5 near the5′ boundary ofexon 6 having the sequence

GCCGAGATAC AATCTTGGTG SEQ. ID. NO: 9and a 3′ mismatch primer which was located in exon 6 near the Asn291Sermutation. The mismatch primer had the sequence

CTGCTTCTTT TGGCTCTGAC TGTA SEQ. ID. NO: 8

PCR amplification reactions were performed using 0.5 μg of genomic DNAin BRL PCR buffer containing 1.5 mM MgCl₂, 200 μM dNTPs, 1 μM eachprimer and 2.5 units Taq polymerase (BRL). The reaction mixture wasdenatured at 95° C. for 1 minute, annealed at 51° C. for 1 minute andextended at 72° C. for 45 seconds for a total of 35 cycles. Twenty μl ofthe PCR product was then digested with 10 units Rsal enzyme, 3.5 μl of10× reaction buffer 1 (BRL), and 9.5 μl of water at 37° C. for 2 hours.The digested fragments were then separated on 2% agarose gel.

Because the combination of the mismatch primer and the Asn291Sermutation produces an Rsal restriction site which is absent when themismatch primer is used to amplify the wild-type gene, the restrictionfragments observed on the agarose gel were different when the mutationwas present. Using this difference as a diagnostic indicator, it wasdetermined that the Asn291Ser mutation was seen in 41 of the 807 or5.09% of the patients in the test group, but in only 3 out of 157 or1.9% of the patients in the control group. When a subgroup of the 494patients in the test group with hypoalphalipoproteinemia was considered,it was found that a higher percentage of these patients, i.e., 6.9% (34out of 494) had the Asn291 Ser mutation. When a further subgroup of thetest group was considered by selecting those individuals with low HDL-Clevels (<1.0%), and excluding those individuals who had bloodglucose>6.8 mmol/l (suggestive of diabetes) and those on β-blockertherapy, 11.3% (12 out of 106 patients) had the mutation. Thisproportion further increased when those with still lower HDL-C levelswere considered separately. Thus, among persons with HDL-C levels lessthan 0.9 mmol/l, 8 out of 68 or 12.5% had the Asn291Ser mutation, whileamong those with HDL-C levels less than 0.8 mmol/l, 5 out of 32 or 15.6%had the Asn291Ser mutation.

Example 9

pRc/CMV vector (Invitrogen) was linearized using XbaI and Hind III. AnXbaI/HindIII fragment containing h-LPL cDNA having a length of about 2.4kb was inserted into the vector. DH5-alpha was transformed with theconstruct. Transformed cells were selected from agar plates based uponampicillin resistance, and grown in LB medium. The plasmid construct,pRc/CMV-hLPL which is shown in FIG. 9, was isolated from the cultures byalkaline lysis and CsCl centrifugation.

Example 10

A purified preparation of an incompetent adenovirus (E1A deletionmutant) was prepared by growing 293 cells in 2 liter spinner flasks to acell density of 4.5×10⁶/ml and infecting the cells with DL312 adenovirusstock at MOI (multiplicity of infection) 20-50 for 1 hour. Forty hourspost infection, the cells were, harvested by centrifugation. A lysatewas prepared by subjecting the harvested cells to 3 freeze/thaw cycles.This lysate was centrifuged in a two-layer CsCl gradient (d=1.25, d=1.4)in a Beckman SW41 swing rotor at 35,000 rpm and 18° C. for 90 minutes.After the ultracentrifugation, the virus was recovered from theinterface between the two CsCl layers using a syringe and a long needle.The recovered virus was then placed onto a CsCl solution (d=1.34) andcentrifuged for 16 hours at 35,000 rpm and 18° C. After thiscentrifugation, the virus was again recovered from the interface and wasthen dialyzed three times (1 hour per cycle) against a sterile buffer(Tris 10 mM, MgCl₂ 1 mM, NaCl 0.135 M). In the third dialysis cycle, thebuffer included 10% glycerol to enhance storage stability. The purifiedvirus was kept frozen at −80° C. until ready to use.

Example 11

Virus prepared as described in Example 10 was mixed with poiylysine (10mM) and EDC (2 mM) for 4 hours at 4° C. in HBS/buffered saline to formadenovirus-polylysine conjugates. The conjugates were re-isolated byCsCl gradient centrifugation using the same protocol as the finalcentrifugation in Example 3.

The re-isolated conjugates (5×10⁹/ml) were incubated with 60-70%confluent Chinese Hamster Ovary cells (CHO K-1) in 2% FBS medium (1 ml)and 6 μg of the plasmid pRc/CMV-hLPL. As a control to assess the extentto which transfection occurred, a second set of samples was prepared inthe same manner using the plasmid pRc/CMV-B-gal which includes a geneencoding β-galactosidase in place of h-LPL. After two hours, the mediumcontaining the conjugates was aspirated out, and new medium (10% FBS)was added to the cells.

By incubating the control cells infected with pRc/CMV-B-gal in thepresence of X-gal, and counting the number of cells which evidenced thecharacteristic blue color which result from cleavage of X-gal byβ-galactosidase, it was determined that the transfection efficiency inthis system varied from 2% when the virus solution was diluted 2000× to50% when the virus solution was diluted 125×. Thus, 50% transfectionefficiency could be achieved in vitro at titers of 0.5-1×10⁸, which isat least 10-fold less than the titers which would normally be used invivo.

To determine the expression of LPL in cells transfected withpRc/CMV-LPL, the activity of LPL was determined and compared to theactivity observed for control cells transfected with pRc/CMV-B-gal. Forthe control cells, the activity measured was 12 mU/ml. For the cellstransfected with pRc/CMV-LPL, the activity measured was 20 mU/ml.

Example 12

The experiments described in Example 11 were repeated, except that thecells used were LPL-deficient cat fibroblast cells or HepG-2 livercells. Table 3 shows the infection efficiencies at various virusdilutions which were determined for these cell types as well as the CHOK-1 cells.

TABLE 3 DILUTION VIRUS 2000X 1000X 500X 250X 125X ECHO K-1 2 5 15 30 50Cat Fibroblast 10 20 50 100 100 HepG-2 20 50 100 100 100

Table 4 shows the LPL activity measured for Cat fibroblast cells, andthe LPL mass measured for cat fibroblast cells and HepG-2 cells. Inaddition, Table 4 shows positive control results for COS EV101 cellswhich are over producers of LPL. It can be seen from this data thatthere is a substantial increase in the plasmid activity and also in theamount of the active dimer form of the enzyme.

TABLE 4 LPL Activity LPL MASS (ng/ml) Cell Type plasmid (mU/ml) totalmonomer dimer CHO K-1 control 12 n.d. n.d. n.d. pRc/CMV-LPL 20 n.d. n.d.n.d. Cat control 0.15 26 24 2 Fibroblasts pRc/CMV-LPL 1.5 128 88 34HepG-2 control n.d. 33 28 6 pRc/CMV-LPL n.d. 164 113 51.5 COS EV101 50530 87 443

Example 13

Vectors for introducing human LDL cDNA into mammalian cells were madeusing the murine leukemia retroviral backbones M3neo, M5neo and JZen1which contain long terminal repeat (LTR) regulatory sequences for themyeloproliferative sarcoma virus. To generate the vectors M3neoLPL andM5neoLPL, a 1.56 kb DraI-EcoRI fragment encompassing the entire LPLamino acid coding region was subcloned into a unique BamHI site located3′ or 5′ to the neomycin phosphotransferase (neo^(f)), respectively.Expression of both genes is LTR driven in these vectors; in M3neoLPL,functional LPL message would derive from the spliced proviraltranscripts whereas for M5neoLPL, LPL message would derive from the fulllength unspliced proviral transcript. To construct JZenLPLtkneo, a 1092bp Xho I/SalI fragment for neo′ was isolated from pMClneo and insertedinto the SalI site of the plasmid pTZ19R, containing the herpes simplexvirus thymidine kinase (tk) promoter. The SmaI/HindIII tkneo fragmentfrom the pTZ19R was inserted into the Hpa I/Hind III site of JZen1. A1.56 kb human LPL cDNA sub-fragment was then cloned in the BamHI site ofJZentkneo. Human LPL cDNA was also subcloned directly into JZen1 toconstruct JZenLPL.

Virus producer cells lines were then made for each of the viralconstructs using the amphotropic retroviral packaging cell line GP-Am12and the esotropic packaging line GP-E86. Both cell lines were culturedin HXM medium, which is Dulbecco's modified Eagle's medium (DME)supplemented with 10% heat-inactivated (55° C. for 20 minutes) newborncalf serum (Gibco-BRL), hypoxanthine (15 μg/ml), xanthine (250 μg/ml)and mycophenolic acid (25 μg/ml). For GP-AM12 cells, hygromycin B (200μg/ml) was also added to the HXM medium. All cells were cultured at 37°C. in a humidified atmosphere of 5% CO₂.

Example 14

A variety of hematopoietic cell lines were tested using the neomycinresistance marker incorporated in the vector to determine whethertransduction occurred as a result of coincubation with M3neoLPL invitro. K562 erythroid cells, HL60 myeloid cells, and U937 and THP-1monocytic cells obtained from the American Type Culture Collection weregrown in RPMI 1640 medium containing 10% fetal bovine serum. The cellswere then infected by cocultivation (24-48 hours) with irradiated (15 Gyx-ray) near confluent producer cells with polybrene 4 μg/ml added to theco-cultivation medium (RPMI/10% fetal bovine serum). After the infectionperiod, the hematopoietic target cells were maintained in suspensionculture for 24 houribefore selection in 1 mg/ml G418. The gene transferefficiencies observed are summarized in Table 5.

The mass of LPL produced was determined for each of the transducedhematopoietic cells lines using two ELISAs. The antibodies used were MAb5D2 which binds to the bioactive dimeric form of LPL and MAb 5F9 whichbinds to both the bioactive dimer and the inactive monomeric form ofLPL. The results are summarized in Table 5. Finally media supernatantswere measured for LPL bioactivity. The results of this study are alsoreported in Table 5.

TABLE 5 Gene Transfer Increase in Increase in Cell Line EfficiencyBioactivity LPL Dimer K562 57% 11-fold 5-fold HL60 47%  9-fold 3-foldU937 45% 14-fold 54-fold  THP-1 41%  4-fold 2-foldThese results demonstrate that for each cell type, good transductionefficiencies were achieved, and production of functional LPL resulted.

Transduced HL60 and THP-01 cells were differentiated in macrophages byexposing the cells to 10 ng/ml of phorbal ester, PdBU (Phorbol12,13-dibutyrate) for 5 days. For HL60 cells, the LPL bioactivityincreased a further 1.8-fold, while the amount of LPL dimer increasedanother 1.8-fold. No further increase was observed upon differentiationof THP-1 cells.

Example 15

NIH 3T3 murine fibroblasts were grown in DME medium containing 10%(vol/vol) fetal bovine serum. The medium on near confluent 60 mm tissueculture plates of viral producer cells 24 hours prior to the plannedinfection with 10 ml DME/10% newborn calf serum. This medium was removedat the time of infection, concentrated 10-fold to a 1.0 ml final volumeby filter centrifugation in Centiprep-30 tubes (Amicon) and diluted 1:4with DME/10% fetal bovine serum with 4 μg/ml polybrene added.Fibroblasts were added to this preparation and incubated for 24-48 hoursat 37° C. 24 hours after viral exposure, cells were subjected toselection in 1.0 mg/ml G418 and grown to confluence. Testing for LPLproduction revealed a 16-fold increase in total LPL production aboveconstitutive levels which consisted almost entirely of dimeric protein,and a 10-fold increase in secreted LPL bioactivity.

Example 16

The experiment of Example 15 was repeated using primary human fibroblastcells, FC 1898 and FC 1901 from diagnostic skin biopsies. No measurablelevels of endogenous LPL protein mass or bioactivity could be detectedprior to retroviral-mediated LPL gene delivery. Post transduction levelsof total LPL mass were massively elevated at least 400 times abovenormal. However, at least 82% of this exogenous LPL protein was of theinactive monomeric form. At least a 52-fold (74.8±22/9) increase indimeric LPL production was seen with significantly elevated secretion ofbioactive LPL, approximately 24 times higher (26.9±3.0) than backgroundLPL levels.

1-15. (canceled)
 16. A method for treating a patient with dyslipoproteinaemia comprising administering to the patient via intravenous injection a recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a nucleic acid sequence coding for a biologically active human lipoprotein lipase (LPL), wherein the nucleic acid sequence is operably linked to a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, and wherein the nucleic acid sequence is expressed so as to cause a reduction of lipoprotein in the patient.
 17. A method for treating a patient with hypertriglyceridaemia comprising administering to the patient via intravenous injection a recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a nucleic acid sequence coding for a biologically active human lipoprotein lipase (LPL) wherein the nucleic acid sequence is operably linked to a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, and wherein the nucleic acid sequence is expressed so as to cause a reduction of triglyceride in the patient.
 18. A method for treating a patient with hypercholesterolaemia comprising administering to the patient via intravenous injection a recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a nucleic acid sequence coding for a biologically active human lipoprotein lipase (LPL), wherein the nucleic acid sequence is operably linked to a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, and wherein the nucleic acid sequence is expressed so as to cause a reduction of cholesterol in the patient.
 19. A method for treating a patient with hyperlipidaemia comprising administering to the patient via intravenous injection a recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a nucleic acid sequence coding for a biologically active human lipoprotein lipase (LPL), wherein the nucleic acid sequence is operably linked to a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, and wherein the nucleic acid sequence is expressed so as to cause a reduction of lipid in the patient.
 20. A method for treating a patient with familial hypertriglyceridaemia comprising administering to the patient via intravenous injection a recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a nucleic acid sequence coding for a biologically active human lipoprotein lipase (LPL) wherein the nucleic acid sequence is operably linked to a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, and wherein the nucleic acid sequence is expressed so as to cause a reduction of triglyceride in the patient.
 21. A method for treating a patient with combined familial hyperlipidaemia and postprandial hyperlipidaemia comprising administering to the patient via intravenous injection a recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a nucleic acid sequence coding for a biologically active human lipoprotein lipase (LPL), wherein the nucleic acid sequence is operably linked to a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, and wherein the nucleic acid sequence is expressed so as to cause a reduction of lipid in the patient.
 22. The method of treatment according to claim 16, wherein the adeno-associated virus is administered by direct injection into the patient's portal vein, such that viral infection is targeted to the liver.
 23. A method for preventing or delaying the onset of coronary artery disease in a human individual having lipoprotein lipase enzyme in which a serine residue is present at amino acid 291 in the enzyme, comprising administering to the individual a recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a nucleic acid sequence coding for a replacement lipoprotein lipase enzyme under the control of a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, said replacement lipoprotein lipase enzyme having an asparagine residue as amino acid 291, wherein the replacement lipoprotein lipase enzyme is produced in the individual to provide a functional lipoprotein lipase enzyme.
 24. The method according to claim 23, wherein the adeno-associated virus further comprises a promoter effective to promote expression of the nucleic acid sequence in human cells.
 25. The method according to claim 23, wherein the adeno-associated virus is administered by parenteral injection.
 26. A recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a nucleic acid sequence coding for a biologically active lipoprotein lipase (LPL) under the control of a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter.
 27. The adeno-associated virus according to claim 26, wherein the nucleic acid sequence is placed under the control of a CMV promoter.
 28. The adeno-associated virus according to claim 26, wherein the nucleic acid sequence is a cDNA sequence.
 29. The adeno-associated virus according to claim 26, wherein the nucleic acid sequence codes for human LPL.
 30. The adeno-associated virus according to claim 26, wherein the nucleic acid sequence is placed under the control of an RSV LTR promoter.
 31. A recombinant defective adeno-associated virus, wherein the rep gene, the cap gene, or the rep and cap genes of the adeno-associated virus are deleted and replaced by a cDNA sequence coding for a biologically active lipoprotein lipase (LPL) under the control of a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter.
 32. The adeno-associated virus according to claim 26, wherein the virus further comprises a nucleic acid sequence enabling the lipoprotein lipase to be directed into a pathway of secretion in an infected cell.
 33. The adeno-associated virus according to claim 31, wherein the secretion sequence is the native secretion sequence of lipoprotein lipase.
 34. The adeno-associated virus according to claim 26, wherein the adeno-associated virus lacks the rep gene.
 35. The adeno-associated virus according to claim 26, wherein the adeno-associated virus lacks the cap gene.
 36. The adeno-associated virus according to claim 26, wherein the adeno-associated virus lacks the rep and cap genes.
 37. A composition comprising the adeno-associated virus according to claim 26 and a pharmaceutically acceptable vehicle.
 38. The composition according to claim 36, wherein the composition is in an injectable form.
 39. A mammalian cell infected in vitro with the adeno-associated virus according to claim 26, whereby a biologically active lipoprotein lipase is expressed from the adeno-associated virus.
 40. The mammalian cell according to claim 38, wherein the cell is a human cell.
 41. The mammalian cell according to claim 39, wherein the cell is selected from the group consisting of a fibroblast, myoblast, hepatocyte, endothelial cell, glial cell, and keratinoctyte.
 42. A composition comprising the infected mammalian cell according to claim 38 and an extracellular matrix.
 43. The composition according to claim 41, wherein the extracellular matrix comprises a gelling compound selected from the group consisting of collagen, gelatin, glycosaminoglycans, fibronectin, and lectins.
 44. The composition according to claim 42, wherein the extracellular matrix comprises a support permitting anchorage of the infected cell.
 45. The composition according to claim 43, wherein the support comprises polytetrafluoroethylene fibers. 